- Email: [email protected]
Improved cryopreservation of bone marrow treated with gold salts
330
ANNUAL
MEETING
heavy (young) enriched platelet suspensions. (Supported by research grants from NIH (HL-09011) and Union Carbide Corporation.) Mechanical Resistance to Volume Reduction in Supercooled Blood Platelets. R. A. KAHN, AND H. T. MERYMAN (American Red Cross Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, MD 20014).
77.
We have previously shown that platelets exposed to hypertonic sodium chloride at 37°C decrease predictably in volume until the osmolality of the extracellular solution reaches four times isotonic. At higher osmolalities volume increases and the platelets are irreversibly injured. If platelets are precooled to -5°C and then exposed to hypertonic salt their volume also decreases, but the rate and extent of reduction is not as great as it was with the 37°C platelets and injury does not occur until eight times isotonic. When platelets at -5°C were ,exposed to a hypertonic solution and then returned to a near isotonic environment the volume reduction was found not to be associated with an influx of extracellular solute as we previously postulated. Furthermore, measurements of the intracellular and extracellular osmolality of platelets in a hypertonic environment revealed a considerable discrepancy between the two compartments with the intracellular osmolality as much as 650 mosm less than the extracellular suspending medium. These results are compatible with a mechanical resistance to volume reduction in supercoo)ed platelets. (Supported in part by National Institutes of Health Contract NIH-‘70-2005.) 78. A Preliminary Evaluation of Additives for the Cryopreservation of Mouse Marrow. .K. R. DILLER, AND H. M. PYLEI (Cryogenic Engineering Laboratory, Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 and Blood Research Institute, Boston, MA 02115). A number of additive compounds have been screened for their ability to protect mouse marrow from freezing injury. Parameters investigated include the composition and concentration of the additive and the cooling rate. Two techniques were employed for freezing the cells: marrow suspensions were frozen at accurately controlled cooling rates either in 2-~1 vol between glass coverslips on a cryomicroscope or in S-ml vol in a small, refrigerated dewar. Recovery was assayed for specimens frozen on the cryomicroscope by postfreeze and postthaw cell morphology and for specimens frozen in the dewnr by microscopic counting, supra-
ABSTRACTS
vital staining, electronic analysis of cell size and distribution, and the stem cell colony technique of Till and McCulloch. The cooling rate was measured via a thermocouple placed into the cell suspension. Survival signatures have been generated in this manner for a number of additives. Some general trends can be observed from this data, including the following: nonpenetrating additives function most effectively at higher cooling rates; penetrating additives function most effectively at lower cooling rates; and as the additive concentration is increased, the best recovery occurs at lower cooling rates. (Supported in part by Research Grants from the American Cancer Society and the Damon Runyon Cancer Fund.) 79. Improved Cryopreservation of Bone Mal.row Treated with Gold Salts. M. D. PERSIDSKY (The
Institute
of Medical
Sciences, Pacific Medical
Center, San Francisco, CA).
In our previous study [Persidsky, Cryobiology 8, 482, (1971)] we have shown that inactivation of lysosomal enzymes by in viva treatment with trypan blue significantly increases bone marrow cell recovery after cryopreservation. In the present study, we have investigated in vitro effects of other inhibitors of lysosomal hydrolysis on cryopreservation of rat bone marrow cells. Cells were incubated at 0°C for 45 min with aurothiomalate (Myochrysine) (0.3 mg/ml) in Hanks’ medium containing 20% rat serum. Thereafter, cells were centrifuged, resuspended in Hanks’ medium containing 10% DMSO, equilibrated for 10 min at O”C, frozen according to the Polge procedure. and then thawed rapidly. The criterion of cell viability was incorporation of [1-“CJglycine at 37°C. The controls were cells which were stored without freezing at 0°C in Hanks’ medium containing 20% serum. Cells frozen without DMSO and without gold incorporated less than 0.4% of the radioactivity of the controls. Cells frozen with DMSO and without the gold pretreatment incorporated 35% of the control level. Cells frozen with DMSO after the gold pretreatment incorporated the same amount of label as the controls. Aurothioglucose and suramin have also been tested. The effects of these compounds have been tested, in addition, on cryopreservation of human white blood cells, and these results will be presented. These findings support our hypothesis that lysosomes are primary targets of cell cryoinjury, and that the inactivation of lysosomal enzymes may be a significant factor in the development of methods for long-term banking of cells and organs by cryopreservation and by perfusion. (Supported by U.S.P.H.S. Grant No. 1 R01 (X12754-01.)
ANNUAL
MEETING
heavy (young) enriched platelet suspensions. (Supported by research grants from NIH (HL-09011) and Union Carbide Corporation.) Mechanical Resistance to Volume Reduction in Supercooled Blood Platelets. R. A. KAHN, AND H. T. MERYMAN (American Red Cross Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, MD 20014).
77.
We have previously shown that platelets exposed to hypertonic sodium chloride at 37°C decrease predictably in volume until the osmolality of the extracellular solution reaches four times isotonic. At higher osmolalities volume increases and the platelets are irreversibly injured. If platelets are precooled to -5°C and then exposed to hypertonic salt their volume also decreases, but the rate and extent of reduction is not as great as it was with the 37°C platelets and injury does not occur until eight times isotonic. When platelets at -5°C were ,exposed to a hypertonic solution and then returned to a near isotonic environment the volume reduction was found not to be associated with an influx of extracellular solute as we previously postulated. Furthermore, measurements of the intracellular and extracellular osmolality of platelets in a hypertonic environment revealed a considerable discrepancy between the two compartments with the intracellular osmolality as much as 650 mosm less than the extracellular suspending medium. These results are compatible with a mechanical resistance to volume reduction in supercoo)ed platelets. (Supported in part by National Institutes of Health Contract NIH-‘70-2005.) 78. A Preliminary Evaluation of Additives for the Cryopreservation of Mouse Marrow. .K. R. DILLER, AND H. M. PYLEI (Cryogenic Engineering Laboratory, Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 and Blood Research Institute, Boston, MA 02115). A number of additive compounds have been screened for their ability to protect mouse marrow from freezing injury. Parameters investigated include the composition and concentration of the additive and the cooling rate. Two techniques were employed for freezing the cells: marrow suspensions were frozen at accurately controlled cooling rates either in 2-~1 vol between glass coverslips on a cryomicroscope or in S-ml vol in a small, refrigerated dewar. Recovery was assayed for specimens frozen on the cryomicroscope by postfreeze and postthaw cell morphology and for specimens frozen in the dewnr by microscopic counting, supra-
ABSTRACTS
vital staining, electronic analysis of cell size and distribution, and the stem cell colony technique of Till and McCulloch. The cooling rate was measured via a thermocouple placed into the cell suspension. Survival signatures have been generated in this manner for a number of additives. Some general trends can be observed from this data, including the following: nonpenetrating additives function most effectively at higher cooling rates; penetrating additives function most effectively at lower cooling rates; and as the additive concentration is increased, the best recovery occurs at lower cooling rates. (Supported in part by Research Grants from the American Cancer Society and the Damon Runyon Cancer Fund.) 79. Improved Cryopreservation of Bone Mal.row Treated with Gold Salts. M. D. PERSIDSKY (The
Institute
of Medical
Sciences, Pacific Medical
Center, San Francisco, CA).
In our previous study [Persidsky, Cryobiology 8, 482, (1971)] we have shown that inactivation of lysosomal enzymes by in viva treatment with trypan blue significantly increases bone marrow cell recovery after cryopreservation. In the present study, we have investigated in vitro effects of other inhibitors of lysosomal hydrolysis on cryopreservation of rat bone marrow cells. Cells were incubated at 0°C for 45 min with aurothiomalate (Myochrysine) (0.3 mg/ml) in Hanks’ medium containing 20% rat serum. Thereafter, cells were centrifuged, resuspended in Hanks’ medium containing 10% DMSO, equilibrated for 10 min at O”C, frozen according to the Polge procedure. and then thawed rapidly. The criterion of cell viability was incorporation of [1-“CJglycine at 37°C. The controls were cells which were stored without freezing at 0°C in Hanks’ medium containing 20% serum. Cells frozen without DMSO and without gold incorporated less than 0.4% of the radioactivity of the controls. Cells frozen with DMSO and without the gold pretreatment incorporated 35% of the control level. Cells frozen with DMSO after the gold pretreatment incorporated the same amount of label as the controls. Aurothioglucose and suramin have also been tested. The effects of these compounds have been tested, in addition, on cryopreservation of human white blood cells, and these results will be presented. These findings support our hypothesis that lysosomes are primary targets of cell cryoinjury, and that the inactivation of lysosomal enzymes may be a significant factor in the development of methods for long-term banking of cells and organs by cryopreservation and by perfusion. (Supported by U.S.P.H.S. Grant No. 1 R01 (X12754-01.)